The plasma membrane of a cell separates the cytoplasm of the cell from its outer environment, and it is primarily composed of a phospholipid bilayer and proteins embedded in the bilayer or attached to the surface thereof. Normally the plasma membrane functions as a gatekeeper which controls trafficking of essential substances into and out of the cell. However, the cell plasma membrane also functions as a permeability barrier and blocks the passage of many useful therapeutic agents. Generally, hydrophilic molecules, highly charged molecules and macromolecules such as nucleic acid, or genes meet difficulties in crossing the cell membranes. Therefore, there is a need for a reliable means of transporting drugs and macromolecules into cells.
Heretofore, a number of transporter molecules such as lipids, polymers, and dendrimers have been proposed to be capable of escorting target molecules across biological membranes, but because they are not water soluble or biodegradable, they tend to precipitate in the cell to cause toxicity.
Proteins having a PTD (protein transduction domain) that allows protein permeation through the plasma membrane include HIV-1 Tat peptide, Antennapedia (Antp) homeodomain protein, Herpes virus protein VP22, and Nuclear localization signal (NLS) sequence.
The above-mentioned protein domain seems to facilitate the permeation across biological membranes without the help of any specific transporter or receptor associated with the cell. Further, they contain a high content of basic amino acid residues such as arginine and lysine. For example, the basic region (i.e., 49-57 a.a.) of the Tat protein, which is a necessary transacting transcriptional activator of HIV virus reproduction, has been reported to play a critical role in the process of the protein permeation through the plasma membrane. A number of studies have reported that various oligopeptides having a multiple arginine residues can be used as molecular transporters.
From these studies, it has been found that oligomers having eight to nine arginine residues show the highest permeability and are most effective in enhancing the transportation of molecules attached thereto across a biological membrane, suggesting that the guanidine group of arginine plays a crucial role in the transportation of molecules attached thereto across a biological membrane.
Wender et al. designed peptoid molecular transporters based on the fact that the biological membrane permeability of a peptide largely depends on the number of the guanidine group in the peptide, the length of the linker chain, and the chirality thereof. It was found that an L-arginine nonamer is 20-times more effective in the transportation across a biological membrane than Tat protein (49-57 a.a.), and a D-arginine nonamer was also much more effective in the uptake by Jurkat cells, as was determined using FACS (P. A. Wender, et al., Proc. Natl. Acad. Sci. U.S.A. 97: 13003, 2000). These results suggest that the permeability of peptides having a specific number of guanidine groups is not significantly dependent on by the chirality of the amino acid (U.S. Pat. No. 6,495,663; Korean Patent Laid-Open Publication No. 2001-12809).
However, such polyarginine peptide or related peptoid molecules have the problems of being eliminated by rapid metabolism in the liver and kidney and the tendency that they show in vivo toxicity. Further, a peptide or peptoid having a plurality of guanidine residues can maintain its helical structure only in a basic environment, and this fact suggests that its membrane permeability depends largely on the positively charged guanidinium groups rather then the secondary or tertiary structure thereof.
The present inventors have therefore endeavored to develop molecular transporters prepared by introducing positively charged guanidinium groups to sugar or its analogue having a linear or branched form with a high density of functionality, and have found that such molecular transporters significantly enhance the transportation of various physiologically active molecules attached thereto either covalently or ionically, across a biological membrane.